Effects of cage vs. net-floor mixed rearing system on goose spleen histomorphology and gene expression profiles

Due to the demands for both environmental protection and modernization of the goose industry in China, the traditional goose waterside rearing systems have been gradually transitioning to the modern intensive dryland rearing ones, such as the net-floor mixed rearing system (MRS) and cage rearing system (CRS). However, the goose immune responses to different dryland rearing systems remain poorly understood. This study aimed to investigate and compare the age-dependent effects of MRS and CRS on the splenic histomorphological characteristics and immune-related genes expression profiles among three economically important goose breeds, including Sichuan White goose (SW), Gang goose (GE), and Landes goose (LD). Morphological analysis revealed that the splenic weight and organ index of SW were higher under CRS than under MRS (p < 0.05). Histological observations showed that for SW and LD, the splenic corpuscle diameter and area as well as trabecular artery diameter were larger under MRS than under CRS at 30 or 43 weeks of age (p < 0.05), while the splenic red pulp area of GE was larger under CRS than under MRS at 43 weeks of age (p < 0.05). Besides, at 43 weeks of age, higher mRNA expression levels of NGF, SPI1, and VEGFA in spleens of SW were observed under MRS than under CRS (p < 0.05), while higher levels of HSPA2 and NGF in spleens of LD were observed under MRS than under CRS (p < 0.05). For GE, there were higher mRNA expression levels of MYD88 in spleens under CRS at 30 weeks of age (p < 0.05). Moreover, our correlation analysis showed that there appeared to be more pronounced positive associations between the splenic histological parameters and expression levels of several key immune-related genes under MRS than under CRS. Therefore, it is speculated that the geese reared under MRS might exhibit enhanced immune functions than those under CRS, particularly for SW and LD. Although these phenotypic differences are assumed to be associated with the age-dependent differential expression profiles of HSPA2, MYD88, NGF, SPI1, and VEGFA in the goose spleen, the underlying regulatory mechanisms await further investigations

Prolactin and its receptor in the follicular hierarchy of chickens

In galliformes, prolactin (PRL) is thought to have both pro- and anti-gonadal effects on steroidogenesis by ovarian follicles dependent on its concentration. However, the role of the major isoform (glycosylated PRL, G-PRL) is unknown. Therefore, a comparison between non-glycosylated (NG-) and G-PRL on steroidogenesis in chicken different size class follicles was carried out. Contrary to the observations in mammals where G-PRL generally shows lower bioactivity than NG-PRL, both isoforms affected both basal and gonadotrophin-stimulated steroidogenesis over a broad concentration range. In granulosa cells of either preovulatory F3-F1 or prehierarchical 6-8 mm follicles, lower levels of NG-PRL stimulated basal estradiol (E2) and progesterone (P4) secretion but at higher levels this effect was reduced. However, G-PRL inhibited basal E2 and P4 secretion in a dose-dependent manner in prehierarchical granulosa cells in contrast to an inducible effect in preovulatory granulosa cells. In accordance with the data for steroids, levels of StAR, CYP11A1, CYP19A1 and 3β-HSD transcripts were up- or down-regulated by both isoforms. Furthermore, G-PRL was less potent than NG-PRL in suppressing FSH- or LH-induced E2 and P4 secretion in preovulatory granulosa cells, whereas G-PRL reduced but NG-PRL enhanced FSH-induced P4 secretion in prehierarchical granulosa cells. In the population of prehierarchical < 6 mm follicles, NG-PRL had a greater effect on suppressing FSH-induced E2 secretion than G-PRL, whereas G-PRL was more effective than NG-PRL in suppressing FSH-induced P4 production. In contrast, with the exception of the synergistic actions of LH and G-PRL on P4 production in the 4-6 mm follicles, both isoforms reduced LH-stimulated E2 and P4 production in all < 6 mm follicles. Thus, glycosylation of PRL can differentially modify its actions on basal and gonadotropin-stimulated steroidogenesis depending on the concentration, the type of gonadotropin (FSH or LH) and the stage of follicle development. Consistent with the effects of PRL on steroidogenesis, the PRL receptor (PRLR) was widely distributed in the follicular hierarchy, with the highest expression in the < 2 mm follicles and a progressive decline as the follicles matured. FSH had the greatest stimulatory effect on expression of PRLR in prehierarchical granulosa cells but this effect declined with follicle maturation, whereas LH showed no effects in all follicular classes examined. NG- and G-PRL were observed to have differential effects on basal and FSH-mediated PRLR expression according to the concentration and the stage of follicle development. As a newly identified ligand for the PRLR in non-mammalian vertebrates including chickens, PRL-like protein (PRL-L) had a similar expression pattern to PRLR during follicle development, with maximal expression in the < 2 mm follicles. Furthermore, PRL-L was more abundant in prehierarchical than preovulatory granulosa cells. FSH increased while LH did not affect expression of PRL-L in prehierarchical granulosa cells. Basal and FSH-induced PRL-L expression were differentially regulated by either isoform of PRL depending on the concentration. Therefore, it is suggested that the differential actions of PRL isoforms on follicular cell steroidogenesis are mediated through modulating the expression of PRLR and PRL-L. By using activators and/or inhibitors, we further demonstrated that in prehierarchical granulosa cells, multiple signaling pathways including PKA, PKC, PI3K-Akt-mTOR and AMPK were not only directly involved in but they could also converge to modulate ERK2 activity to regulate FSH-induced PRLR and PRL-L expression.Chez les galliformes, c'est pensé que la prolactine (PRL) a des effets pro- et anti-gonadiques sur la stéroïdogenèse par les follicules ovariens en fonction de sa concentration. Cependant, le rôle de l'isoforme majeure (PRL glycosylée, G-PRL) est inconnu. Par conséquent, une comparaison entre la prolactine non glycosylée (NG-) et G-PRL sur la stéroïdogenèse dans des follicules de différentes classes de taille de poulet a été effectuée. Contrairement aux observations chez les mammifères où la G-PRL présente généralement une bioactivité plus faible que la NG-PRL, les deux isoformes ont affecté la stéroïdogenèse basale et aussi stimulée par la gonadotrophine à nombreuses concentrations. Dans les cellules de la granulosa des follicules préovulatoires F3-F1 ou préhérarchiques de 6 à 8 mm, des niveaux inférieurs de NG-PRL ont stimulé la sécrétion basale d'estradiol (E2) et de progestérone (P4), mais à des niveaux plus élevés cet effet a été réduit. Cependant, la G-PRL a inhibé la sécrétion basale d'E2 et de P4 de manière dose-dépendante dans des cellules de granulosa préhérarchiques, contrairement à un effet inducible dans des cellules de granulosa préovulatoires. Conformément aux enregistrements sur les stéroïdes, les niveaux des transcrits StAR, CYP11A1, CYP19A1 et 3ß-HSD ont été régulés à la hausse ou à la baisse par les deux isoformes. En outre, la G-PRL était moins puissante que la NG-PRL dans la suppression de la sécrétion d'E2 et P4 induite par la FSH ou la LH dans les cellules de granulosa préovulatoires, tandis que la G-PRL réduit mais la NG-PRL augmentait la sécrétion de P4 induite par la FSH dans les cellules granulosa préhérarchiques. Dans la population de follicules préhérarchiques < 6 mm, la NG-PRL a eu un effet plus important sur la suppression de la sécrétion d'E2 induite par la FSH que la G-PRL, tandis que la G-PRL était plus efficace que la NG-PRL pour inhiber la production de P4 induite par la FSH. Contrairement, à l'exception des actions synergiques de LH et de G-PRL sur la production de P4 dans les follicules de 4-6 mm, les deux isoformes ont réduit la production d'E2 et P4 stimulée par LH dans tous les follicules < 6 mm. Conformément aux effets de la PRL sur la stéroïdogenèse, le récepteur PRL (PRLR) a été largement distribué dans la hiérarchie folliculaire, avec la plus haute expression dans les follicules de < 2 mm et un déclin progressif à mesure que les follicules se développaient. La FSH a eu le plus grand effet stimulant sur l'expression du PRLR dans les cellules de la granulosa préhérarchique, mais cet effet a diminué avec la maturation des follicules, alors que la LH n'a montré aucun effet dans toutes les classes folliculaires examinées. On a observé que NG- et G-PRL ont des effets différentiels sur l'expression du PRLR basal et médiée par la FSH en fonction de la concentration et du stade de développement du follicule. En tant que ligand nouvellement identifié pour le PRLR chez des vertébrés non mammifères comprenant des poulets, la protéine PRL-like (PRL-L) présentait un profil d'expression similaire au PRLR pendant le développement du follicule, avec une expression maximale dans les follicules < 2 mm. De plus, le PRL-L était plus abondant dans les cellules préhérarchiques que dans les granulosa préovulatoires. La FSH a augmenté tandis que la LH n'a pas affecté l'expression de PRL-L dans les cellules granulosa préhérarchiques. L'expression de PRL-L basale et induite par la FSH était différentiellement régulée par l'isoforme de la PRL selon la concentration. En utilisant des activateurs et / ou des inhibiteurs, on a également démontré que dans des cellules de granulosa préhérarchiques, des voies de signalisation multiples comprenant PKA, PKC, PI3K-Akt-mTOR et AMPK étaient non seulement directement impliquées mais elles pourraient également converger pour moduler l'activité de ERK2 pour réguler l'expression de PRLR et PRL-L induite par FSH

Expression pattern of <i>PRLR</i> mRNA in chicken developing follicles.

<p>(A) Abundance of <i>PRLR</i> mRNA in the stroma and walls of prehierarchical (< 2, 2–4, 4–6 and 6–8 mm) and preovulatory (9–12 mm, F5 and F4) follicles. (B) Relative <i>PRLR</i> mRNA levels in theca and granulosa cell layers isolated from the three largest preovulatory follicles (namely F3-F1; F3 < F2 < F1). Relative expression level was normalized to <i>18S rRNA</i>. Data are expressed as fold differences ± SEM compared to either the stroma or F3 granulosa cells (n = 4 hens). Different lowercase letters indicate a significant effect of follicular developmental stage, whilst different uppercase letters indicate a significant effect of cell type (granulosa versus theca cell layer). <i>P</i> < 0.05 was accepted as statistically significant.</p

Role of PI3K-Akt-mTOR and AMPK signaling pathways in FSH-induced <i>PRLR</i> and <i>StAR</i> mRNAs expression in cultured granulosa cells of prehierarchical (6–8 mm) follicles.

<p>(A and C) Changes in relative mRNA levels of <i>PRLR</i> (A) and <i>StAR</i> (C) after culture of granulosa cells for 4 h in the absence or presence of 10 ng/ml FSH together with PI3K inhibitor (20 μM LY294002) or mTOR inhibitor (10 μM Rapamycin). (B and D) Relative <i>PRLR</i> (B) and <i>StAR</i> (D) mRNAs levels in granulosa cells cultured for 4 h in the absence or presence of 10 ng/ml FSH, or together with AMPK activator (1 mM AICAR). Relative expression level was normalized to <i>18S rRNA</i>. Data are expressed as fold differences ± SEM of three independent experiments using tissues from different hens and are compared to control cells. ^*, <i>P</i> < 0.05 compared to control cells; ^#, <i>P</i> < 0.05 compared to cells only stimulated by 10 ng/ml FSH.</p

Role of PKA and PKC signaling pathways in FSH-induced <i>PRLR</i> and <i>StAR</i> mRNAs expression in cultured granulosa cells of prehierarchical (6–8 mm) follicles.

<p>(A and C) Changes in relative mRNA levels of <i>PRLR</i> (A) and <i>StAR</i> (C) after culture of granulosa cells for 4 h in the absence or presence of 10 ng/ml FSH in combination with PKA activator (10 μM Forskolin) or inhibitor (20 μM H89). (B and D) Changes in relative mRNA levels of <i>PRLR</i> (B) and <i>StAR</i> (D) in granulosa cells treated without or with 10 ng/ml FSH in combination with PKC activator (20 nM PMA) or inhibitor (10 μM GF109203X). Relative expression level was normalized to <i>18S rRNA</i>. Data are expressed as fold differences ± SEM of three independent experiments using tissues from different hens and are compared to control cells. ^*, <i>P</i> < 0.05 compared to control cells; ^#, <i>P</i> < 0.05 compared to cells only stimulated by 10 ng/ml FSH.</p

Effects of manipulation of several intracellular signaling pathways on basal and FSH-induced ERK2 phosphorylation in cultured granulosa cells of prehierarchical (6–8 mm) follicles.

<p>Top panel: Representative western blot analysis of phosphorylated and total ERK2 in granulosa cells treated without or with 10 ng/ml FSH in combination with PKA activator or inhibitor (10 μM Forskolin or 20 μM H89, respectively), or PKC activator or inhibitor (20 nM PMA or 10 μM GF109203X, respectively), or PI3K inhibitor (20 μM LY294002) or mTOR inhibitor (10 μM Rapamycin), or AMPK activator (1 mM AICAR). β-actin was used as loading control. Bottom panel: Quantitative analysis of relative protein abundance of phosphorylated ERK2 by densitometry using Image Lab software (Version 4.1, Bio-Rad laboratories). Relative protein abundance was normalized to total ERK2. Data are expressed as fold differences ± SEM of three independent experiments using tissues from different hens and are compared to control cells. ^*, <i>P</i> < 0.05 compared to control cells; ^#, <i>P</i> < 0.05 compared to cells only stimulated by 10 ng/ml FSH.</p

Abundance of ERK2 protein in chicken developing follicles and its role in FSH-induced <i>PRLR</i> and <i>StAR</i> expression in cultured granulosa cells of 6–8 mm follicles.

<p>(A) Top panel: Representative western blot analysis of phosphorylated and total ERK2 in the stroma and walls of prehierarchical (< 2, 2–4 and 4–6 mm) follicles as well as in theca and granulosa cell layers separated from the 6–8 mm and F3-F1 follicles. β-actin was used as loading control. Bottom panel: Quantitative analysis of relative protein abundance of phosphorylated ERK2 by densitometry using Image Lab software (Version 4.1, Bio-Rad laboratories). Relative protein abundance was normalized to total ERK2. Data are expressed as fold differences ± SEM compared to an appropriate tissue (n = 4 hens). Bars with different letters are significantly different at <i>P</i> < 0.05. (B and C) Changes in relative mRNAs levels of <i>PRLR</i> (B) and <i>StAR</i> (C) in granulosa cells treated without or with 10 ng/ml FSH, or together with the MEK inhibitor (1 μM PD0325901) after a 4-h culture. Relative mRNA expression level was normalized to <i>18S rRNA</i>. Data are expressed as fold differences ± SEM of three independent experiments using tissues from different hens and are compared to control cells. ^*, <i>P</i> < 0.05 compared to control cells; ^#, <i>P</i> < 0.05 compared to cells only stimulated by 10 ng/ml FSH.</p

Primer pairs for real-time quantitative PCR.

<p>Primer pairs for real-time quantitative PCR.</p

Metabolomic Analysis of SCD during Goose Follicular Development: Implications for Lipid Metabolism

Stearoyl-CoA desaturase (SCD) is known to be an important rate-limiting enzyme in the production of monounsaturated fatty acids (MUFAs). However, the role of this enzyme in goose follicular development is poorly understood. To investigate the metabolic mechanism of SCD during goose follicular development, we observed its expression patterns in vivo and in vitro using quantitative reverse-transcription (qRT)-PCR. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to determine a cellular model of SCD function in granulosa cells (GCs) via SCD overexpression and knockdown. qRT-PCR analysis showed that SCD was abundantly expressed in the GC layer, and was upregulated in preovulatory follicles. Peak expression was found in F1 and prehierarchal follicles with diameters of 4&ndash;6 mm and 8&ndash;10 mm, respectively. We further found that mRNA expression and corresponding enzyme activity occur in a time-dependent oscillation pattern in vitro, beginning on the first day of GC culture. By LC-MS/MS, we identified numerous changes in metabolite activation and developed an overview of multiple metabolic pathways, 10 of which were associated with lipid metabolism and enriched in both the overexpressed and knockdown groups. Finally, we confirmed cholesterol and pantothenol or pantothenate as potential metabolite biomarkers to study SCD-related lipid metabolism in goose GCs